Topical preventative medicament against burns-related systemic inflammatory response syndrome

ABSTRACT

A topical preventative medicament against burns-related systemic inflammatory response syndrome containing a low-molecular-weight chaperone as the main active ingredient.

RELATED APPLICATIONS

Foreign priority benefits are claimed under 35 U.S.C. §119(a)-(d) or 35U.S.C. §365(b) of Japanese application number 2012-143554, filed Jun.26, 2012, the contents of each application which are incorporated byreference herein in their entirety.

TECHNICAL FIELD

The present invention relates to a topical preventative medicamentagainst burns-related systemic inflammatory response syndrome.

BACKGROUND OF INVENTION

If someone suffers a burn at their workplace or at home, for example,the conventional advice is to cool the affected area by holding it underrunning water or similar for the requisite time (e.g. 15 to 30 minutes).If no running water is available nearby, it is desirable to cool theaffected area by applying, for example, a poultice containing a coolingagent (e.g. as described in Patent Reference 1). Particularly if theburn is severe, advice and treatment need then to be sought at a medicalinstitution without delay. Treatment appropriate to the depth of theburn can then be provided by the medical institution.

In the case of severe burns, there are times when simply cooling theaffected area will not necessarily be sufficient as emergency treatmentto be given at the stage before medical advice can be sought at amedical institution.

That is to say, an inflammatory reaction may be elicited by a range ofmediators such as inflammatory cytokines produced and released locallyor systemically when a victim suffers burns, and this may develop into acondition known as systemic inflammatory response syndrome (SIRS). Onecause of SIRS is thought to be the release of cytokines fromimmunological cells against proteins denatured as a result of the burn.

If the victim goes into SIRS, the symptoms may progress rapidly, leadingto septicemia or shock and even death in some cases. In the case ofsevere burns, it is extremely important to take emergency action tosuppress SIRS the same time as applying cooling. The present inventionwas devised in the light of these circumstances and provides a topicalpreventative medicament against burns-related systemic inflammatoryresponse syndrome capable of suppressing burns-related SIRS by beingapplied directly to the affected area when someone has suffered a burn.

SUMMARY OF THE INVENTION

In order to overcome the current problem described above, the topicalpreventative medicament against burns-related systemic inflammatoryresponse syndrome in this invention contains a low-molecular-weightchaperone as its main active ingredient. In addition to the lowmolecular-weight chaperone, the medicament can further contain one ormore of (i) a water-soluble fullerene, (ii) histamine and (iii) abuffer.

According to one aspect of the invention, a topical medicament againstburns-related systemic inflammatory response syndrome is provided. Themedicament contains one or more low-molecular-weight chaperones as themain active ingredient. The chaperone may be any of the molecules orclasses of molecules as described in detail below. In importantembodiments, the low molecular-weight chaperone is a protein with ahomo-oligomeric structure and 9-48 associated subunits with molecularweight of 12-43 kDa. In important embodiments, the low molecular-weightchaperone is a small heat shock protein. In important embodiments, thelow molecular-weight chaperone is αB-crystallin.

The topical preventative medicament, in any of the embodiments asdescribed above, can optionally contain a water-soluble agent forenhancing tissue penetration, such as a water soluble fullerene.

The topical preventative medicament, in any of the embodiments asdescribed above, can optionally histamine.

The topical preventative medicament, in any of the embodiments asdescribed above, can optionally contain a buffer.

According to another aspect of the invention, a method of treating aburn is provided. According to another aspect of the invention, a methodof increasing the survival chances in an animal having a severe burn isprovided. According to another aspect of the invention, a method ofinhibiting the development or progression of SIRS is provided. Accordingto another aspect of the invention, a method is provided for suppressingrises in one or more of TNF-α and white blood cell count followingsevere burns. The methods described in this paragraph involveadministering topically to a subject an effective amount of any one ofthe topical preventative medicaments described above.

According to another aspect of the invention, a pharmaceuticalpreparation is provided. The pharmaceutical preparation comprises achaperone for treating a burn. The treatment can be topical treatment.The chaperone can be any one or more of the chaperones described herein.The pharmaceutical can be any of the topical preventative medicamentsdescribed herein.

According to another aspect of the invention, a medical treatment systemis provide. The system is a bottle or container having a spray nozzleand containing any of the topical preventative medicaments describedherein. In any of the forgoing embodiments the chaperone can be presentat a concentration of 1-100 μM, 10-80 μM or 25-75 μM. In any of theforegoing embodiments, histamine can be present, and present at aconcentration of 0.05-1.0 μM, 0.05-0.5 μM or 05-0.1 μM. In any of theforegoing embodiments, the medicament can be an aqueous bufferedsolution, at 7.5-8.5. In any of the foregoing embodiments, themedicament can contain a buffer present at 10-50 mM or even 20-30 mM. Inany of the foregoing embodiments, the buffer can be phosphate buffer.

Taking the invention of claim 1, there can be provided a topicalpreventative medicament against burns-related systemic inflammatoryresponse syndrome that is capable of treating a burn if applied to theaffected area immediately following a burn because it incorporates alow-molecular-weight chaperone as its main active ingredient. In someembodiments, the medicament suppresses burns-related SIRS. In someembodiments, the medicament ameliorates the side effects associated withone or more of a first degree burn, a second burn and a third degreeburn. In some embodiments, the medicament enhances healing of a burn by,for example, speeding the process of healing or lessening (relative toan untreated burn) the extent of the damage resulting from the burn.

Moreover, in important embodiments, there is provided a topicalpreventative medicament, e.g., against burns-related systemicinflammatory response syndrome, with high levels of pharmaceuticalstability and safety because the low-molecular-weight chaperone isαB-crystallin, which is ubiquitous in almost all systemic tissues of thebody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Is a graph showing the mean survival and standard error at eachpoint for five runs with ten animals (5 sample animals, 5 controls).

FIG. 2 Is a graph showing that survival was about 100% in the grouptreated with topical medicament B that had been mixed with awater-soluble C60 fullerene to improve cell membrane penetrability(p<0.005), and the level was higher than in the group treated withtopical medicament A.

FIG. 3 Is a graph showing test results demonstrating that the level ofserum TNF-α was markedly suppressed by the application of topicalmedicament A compared to no treatment.

FIG. 4 Is a graph showing that the white blood cell count was markedlycontrolled by the application of topical medicament A compared to notreatment.

DETAILED DESCRIPTION

The present invention provides a topical preventative medicament againstburns-related systemic inflammatory response syndrome, containing alow-molecular-weight chaperone as its main active ingredient.

Burns are injuries sustained by the epidermis or subcutaneous tissue dueto heat or chemical substances. In particular, the concept of burns inthe context of this specification includes not only heat burns caused byheat from hot water or hot oils, but also chemical burns caused bychemical products such as acids or alkalis, electric shock burns causedby electric current and the like, and radiation burns caused byhigh-dose radiation.

Burns are classified according to the depth of the injury into first(I), second (II) and third (III) degrees. In first-degree burns,dilatation of the blood vessels causes erythema. In second-degree burns,there is blistering due to vascular hyperpermeability and halting of theblood flow. Third-degree burns involve cutting off of the blood flow andnecrosis.

As a rule, second-degree burns can be treated, but wound closure cannotbe expected in third-degree burns unless a skin graft is applied.Moreover, severity differs depending not only on the depth but also thebreadth of the burn.

The treatment of extensive burns requires fluids to counter shock in theearly stages after the injury, infection control until closure of thewound, nutritional management by central venous or tube feeding, andskin grafts and general surgical interventions to save life. However, insevere cases, the victim's life often cannot be saved despite thesemeasures.

As severity cannot be satisfactorily assessed from burn area alone, theburn index (BI: Body Index) represented by the formula below, whichtakes account of depth and area, is used to assess this.

BI=[area of second-degree burn(%)/2]+area of third-degree burn(%)

Local therapies to treat wounds immediately after a burn occursinclude 1) cooling of the heat wound: as a rule, cooling for at leastthirty minutes under running water, 2) wound treatment: disinfectingwith 0.02-0.5% chlorhexidine and treating with ointments, and 3) specialtreatment: in full-circumference burns to the extremities of thirddegree or higher, the blood flow may be obstructed and compartmentsyndrome develop. If tissue pressure reaches 40 mmHg or more,escharotomy is performed without delay.

Conservative local therapies include 1) closure: azulene ointment,Eksalb, gentamycin sulfate ointment or fradiomycin sulfate dressings areused for shallow burns up to Hs, but for deeper burns of IId or III whenthere is thick eschar and a bacteria phase readily forms beneath it,silver sulfadiazine creams or mafenide acetate creams are used as thesepenetrate well even into thick eschar, 2) water treatment (warm bathtreatment, shower baths) and 3) bandaging.

Moreover, local surgical treatments include 1) debridement (removal ofnecrotic tissue) and 2) skin grafts: carried out on third-degree burnswhen epithelialization cannot be expected. This is usually autologousskin graft. Skin grafts are carried out early on to reduce illnessduration and preserve function. Culturing techniques for graftepithelium have also been improving of late and as this is now evencovered by health insurance, it is starting to be applied even toextensive burns and is contributing to improved survival rates.

However, as the rescue rate is much lower if BI exceeds 100 (J Jpn SurgSoc 85: 739-748, 1985), treatments that will improve rescue rates arebeing sought.

Against this general background, the present inventors noticed thecytoprotective effect of low-molecular-weight chaperones against heattreatment. Following intense research to find agents that would fulfillthe role of novel treatment for severe burns, they discoveredsurprisingly that applying a low-molecular-weight chaperone to theaffected area—something that had never been tried before—had the effectof greatly improving survival rates from severe burns. They devised theinvention of the present application based on these findings.

The present invention relates to a topical preventative medicament totreat burns, and is particularly useful against SIRS in life-threateningsevere burns, characterized in that it contains a low-molecular-weightchaperone as its main active ingredient. Molecular chaperones areproteins that assist the non-covalent folding or unfolding of otherproteins. One major function of chaperones is to prevent newlysynthesized polypeptide chains and assembled subunits from aggregatinginto nonfunctional structures. It is for this reason that manychaperones, but by no means all, are also heat shock proteins. (HSPinitially came from “heat shock”, but later on, involved many otherfactors associated with stresses which induce abnormal conformations;chemical, physical and degenerative like Alzheimer βamyloidosis.)

Molecular chaperones can be divided into two species; large ones whichare 60-90 k dalton (kD) of proteins, eg. HSP60/70/90, and small ones orHSPs (sHSP) which are less than about 30 kD, typically 20-30 kD, egsHSP16.5/20/27, Crystallin aA/aB. At least 10 sHSP (Hsp:B1,B2, B3, B4,B5, B6, B7, B8, B9 and B10) are found in human and αB-crystallin isclassified into 5B among them. sHSP is immediately produced in responseto various stimuli, under control by heat shock factors, caused by notonly high temperature but also under stresses; pH, abnormal pressure,endoplasmic stress (induced by irregular proteins produced in thecellular ribosome). sHSPs trap substrate unfolded/immature substrateproteins (non-native states) by targeting their hydrophobic portions inATP-independent fashion and transfer them to HSP60/70/90. Thus sHSPs areprimary chaperone molecules for cellular metabolic maintenance,replication and apoptosis.

The topical preventative medicament against burns-related systemicinflammatory response syndrome of the present embodiment may obviouslybe used in humans, but may also be used in animals other than humanbeings, including mammals.

Proteins belonging to the class of so-called small heat shock proteins(sHSP) are ideal as low-molecular-weight chaperones to be used in thistopical medicament.

Low-molecular-weight chaperones include proteins with a homo-oligomericstructure and 9-48 associated subunits with molecular weight of 12-43kDa and are present ubiquitously in the living world. Such chaperonescan be characterized by having an α-crystallin domain comprising aboutninety amino acid residues, but they exhibit low sequence homology andthe amino acid sequences enclosed by the N-terminal and C-terminal areextremely diverse.

While not wishing to be bound by any theory, with low-molecular-weightchaperones, it is thought that substrate binding sites open up when thehomo-oligomer is subjected to stress such as heat, exposing richlylipophilic regions and suppressing the irreversible aggregation ofdenatured proteins. In other words, low-molecular-weight chaperones havemolecular activity that serves to prevent the irreversible aggregationof other proteins, but they do not support ATP-dependent refolding suchas seen with Hsp70 or chaperones. Accordingly, it is thought thatsubstrate supplemented by low-molecular-weight chaperone will bereleased from substrate/low-molecular-weight chaperone complexes by theaction of other molecular chaperones such as the Hsp104 and Hsp70/40systems, and will be refolded.

It thus is believed that this topical medicament will control SIRSquickly without the need for ATP as a result of the low-molecular-weightchaperone suppressing the irreversible aggregation of denatured proteinin the affected area, which event otherwise would trigger theimmunological reaction leading to SIRS.

Incidentally, it is desirable for the topical medicament to be in theform of an aqueous fluid. If designed like this, there will be littleirritation of the affected area and penetration of thelow-molecular-weight chaperone into the affected area can take placeefficiently. This will also have the effect of keeping the affected areamoist and preventing drying.

This topical medicament will be extremely useful as an everydaymedicinal product to keep available, for example, in the home orworkplace. In other words, by using it without delay on a severe burnbefore the victim can be seen at a medical center or taken away as anemergency, it will be possible to prevent the onset or worsening of SIRSand improve rescue rates for victims as much as possible.

It is therefore advisable to keep the topical medicament in arefrigerator or similar when not being used with a view to preventingdeterioration due to long-term storage. Storage in a refrigerator isalso convenient in order to cool the affected area when the product isused.

There are no particular restrictions on the method by which this topicalmedicament is used, but one approach would be to use it following thecooling of the affected area under running water described earlier.However, in emergency situations when running water is not available,the topical medicament should be used in conjunction with cooling of theaffected area. That is to say, the topical medicament may be appliedwithout cooling the affected area under running water. In particular,this type of approach would seem to be important in situations whenthere would be concern about the progression of SIRS with cooling forlong periods.

It is also desirable to add the low-molecular-weight chaperone to anaqueous buffer for the topical medicament, so as to keep thelow-molecular-weight chaperone stable in an active state over longperiods, such as for 3 months, 6 months, 9 months, twelve months or evenyears. There are no particular restrictions on the buffer solution to beused as such a medium as long as it can buffer any changes in pH duringstorage, does not hamper the opening of substrate binding sites of theoligomer in order to activate the low-molecular-weight chaperone asdescribed later, and also has no adverse effects on the body or theaffected area (for example, irritation or mutagenicity, etc.).

Examples of such buffers taking well-known materials include phosphatebuffer, buffers using HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) and buffers using MOPS (3-morpholinopropane sulfonicacid).

The concentration of the low-molecular-weight chaperone in the topicalmedicament is, for example, 1-100 μM, preferably 10-80 μM and morepreferably 25-75 μM.

If the concentration of the low-molecular-weight chaperone is less than1 μM, there will be very little low-molecular-weight chaperone per unitvolume and it will be less likely to suppress SIRS. Moreover,concentrations exceeding 100 μM give no marked increase in SIRS controland are undesirable economically. It will be understood, however, thatconcentrations above 100 μM can be used.

The topical medicament can be both economical and provide satisfactorySIRS control by setting the concentration of the low-molecular-weightchaperone to within the above range.

A tissue penetration enhancer can also be added to the topicalmedicament. For example, a water-soluble fullerene may also be added tothe topical medicament. Such fullerenes typically carry charged groupssuch as hydroxyl or amine functionalities. The ability of thelow-molecular-weight chaperone to penetrate cell membranes can beimproved by adding a water-soluble fullerene, and SIRS can be stillbetter suppressed.

The concentration of this added fullerene may be set, for example, at0.5-2% weight per volume and in some embodiments 1-2%. If the fullerenecontent is less than 0.5%, very little advantage in terms of aiding cellmembrane penetration by the low-molecular-weight chaperone is seen.Using more than 2% does not provide any great improvement in cellmembrane penetration by the low-molecular-weight chaperone and isuneconomic. It will be understood, however, that concentrations above 2%can be used. Setting the content of water-soluble fullerene at 0.5-2%and preferably 1-2% is economic and assists penetration of cellmembranes by the low-molecular-weight chaperone.

The topical medicament may also incorporate histamine. Histamine isgenerally believed to aggravate SIRS. However, studies by the inventorsindicated that adding histamine to the topical medicament helps tocontrol the onset of SIRS and contributes to improved survival rates byraising the local penetrability of the low-molecular-weight chaperone.

The concentration of histamine added may be set, for example, at0.05-1.0 μM, preferably at 0.05-0.5 μM and more preferably at 0.05-0.1μM. If the histamine content goes below 0.05 μM, hardly anySIRS-suppressing effect is seen. Levels above 1.0 μM cannot be expectedto provide any marked improvement in SIRS suppression and are noteconomic. It will be understood, however, that concentrations above 1.0μM can be used. Setting the content of the water-soluble histamine inthe topical medicament at 0.05-1.0 μM, preferably at 0.05-0.5 μM andmore preferably at 0.05-0.1 μM is economic and enables theSIRS-suppressing effect to be elicited.

The low-molecular-weight chaperone incorporated into the topicalmedicament can be αB-crystallin.

Crystallins is the general title given to specific water-solubleproteins in the crystalline lens, and their name is derived from thefact that they are transparent proteins. The crystalline lens of man ismade up mainly of the proteins α-, β- and γ-crystallin.

Alpha-crystallin forms large associates through the auto-association ofthe subunit proteins αA- and αB-crystallin, and is understood tomanifest its chaperone function to maintain the transparency of the lensthrough interaction with β- and γ-crystallin. However, no abnormality inlens function has been demonstrated in mice in which αB-crystallin hasbeen knocked out. Besides the lens, it is present also in the heart,skeletal muscle, kidney, lung, CNS glial cells and so on.

AlphaB-crystallin belongs the heat-shock protein (Hsp) family, and inparticular has a high level of homology with the low-molecular-weightHsp27. It therefore is likely that Hsp27 could be used in combinationwith or as a substitute for αB-crystallin. In other words,low-molecular-weight chaperones incorporated in this topical medicamentalso include Hsp27. Put another way, in important embodiments, sHSPswith an α-crystallin domain may be used as the low-molecular-weightchaperone incorporated in the topical medicament.

When αB-crystallin is used as the low-molecular-weight chaperone in thetopical medicament, its stability can be increased by setting the pH ofthe buffer solution mentioned earlier at 7.5-8.5.

However, in order for the αB-crystallin oligomer to break down and beactivated, dissociation of the C-terminal IXI motif from the substratebinding site under the low-pH conditions prevalent at sites ofinflammation is required, and there is a risk that strong buffer mightrather reduce the effect.

The concentration of the buffering ingredient, such as phosphate, HEPESor MOPS should thus be set at a level (buffer capacity) that contributesto the storage stability (stable pH) of the low-molecular-weightchaperone during storage, while ensuring that buffering performance isreadily attenuated or lost (pH change) when the medicament is applied tothe affected area during use. For example, if using phosphate buffer asthe buffer solution, it should be set at 10-50 mM and preferably 20-30mM.

With the other buffer solutions too, the concentration of the bufferingingredient appropriate for the topical medicament may be determined asrequired by checking SIRS suppression in accordance with each of thetest methods outlined below. Opening of substrate binding sites of theoligomer occurs at high temperatures, as indicated by the name ‘heatshock protein’, but it can also take place due to changes in pH as wellas phosphorylation by enzymes. With this topical medicament, the changein pH at the time of use acts as the trigger to promote activation, andconsequently, even though the low-molecular-weight chaperone is in astable state during storage, it can be rapidly changed to active modewhen it is used.

The topical medicament is described in more detail below throughspecific examples.

Examples Preparation of a Topical Preventative Medicament AgainstBurns-Related Systemic Inflammatory Response Syndrome

The preparation of this topical medicament is described first. In thepresent embodiment, reference is made to a topical preventativemedicament against burns-related systemic inflammatory response syndromecontaining αB-crystallin as the low-molecular-weight chaperone, but itgoes without saying that other low-molecular-weight chaperones could beused.

There are no particular restrictions on the processes whereby theαB-crystallin, which is the essence of the present topical medicament,is produced. Recombinant αB-crystallin expressed in E. coli was usedhere. This was prepared as follows.

Firstly, E. coli BL-21 transformed with an His-tagged PET vectorencoding αB-crystallin was incubated overnight in LB medium at 37° C.

Next, isopropyl-β-D-thiogalatoside (IPTG) was added to the medium to afinal concentration of 1 mM and it was further incubated for four hoursat 37° C. so as to induce overexpression of the protein.

The cells were collected by centrifugation (8000×g, 20 mins, 4° C.) andthe supernatant fluid removed. They were then suspended in pH 7.8 lysisbuffer comprising 0.25 g/mL lysozyme, plus 25 mM Tris/50 mM NaCl/0.9%glucose/1 mM EDTA containing a protease inhibitor cocktail (Sigma) andwere solubilized in ice with ultrasound.

The solubilized fraction was separated by centrifugation (10,000×g, 20mins, 4° C.) and the resulting supernatant fluid was dialyzed with 20 mMphosphate buffer, pH 7.8, containing 0.5 M table salt and applied to aNi-chelating column (Pharmacia) equilibrated with the same buffer.

After the column had been flushed with 20 mM imidazole equilibratedbuffer, the fraction was eluted with 20 mM imidazole equilibratedbuffer.

The eluted fraction was then concentrated with Amicon and dialyzed with20 mM phosphate buffer. The resulting fluid was taken as the topicalpreventative medicament against burns-related systemic inflammatoryresponse syndrome (hereafter, topical medicament A) and subjected to thetests described below.

In order to apply the topical preventative medicament againstburns-related systemic inflammatory response syndrome to affected areasin the explanations below, it was decided that 1 mL of a product at aconcentration of 50 μM calculated from the molar extinction coefficientshould be sprayed on having removed LPS (lipopolysaccharides) with ETClean (JNC K.K.).

Confirmation of Mortality Rate Reduction—Test 1

Next, mice that had suffered burns were used in tests to confirmreductions in mortality rate depending on whether or not a topicalpreventative medicament of this embodiment against burns-relatedsystemic inflammatory response syndrome had been applied.

The tests were carried out with reference to a method established as amodel reflecting life-threatening severe burns. This was considered toprovide an ideal evaluation system for tests to confirm reductions inmortality rates using a topical preventative medicament of thisembodiment against burns-related systemic inflammatory responsesyndrome.

Specifically, the fur was shaved from an area 3 cm in diameter on thebacks of ICRSW male mice and depilation was carried out with Veet(Reckitt Benckiser Japan).

Next, 20 mg/kg of pentobarbital was given by the intraperitoneal route,and 25% of the animals' total body surface area (TBSA) with thedepilated area in the center was immersed in hot water at 100° C. foreight seconds. The animals were monitored and mortality rates comparedafter six hours in the group treated with topical medicament A (N=5) andthe untreated group (N=5). The results are shown in FIG. 1.

It is evident from FIG. 1 that whereas survival in the untreated groupwas about 20% over the first ten hours (21.0±14.3%), it was about 75%(75.0±16.6%) in the group treated with topical medicament A. Tests forsignificant difference between the two groups also suggested that thesurvival rate was significantly higher (p=0.0047) as a result ofapplying topical medicament A (applying 50 μM αB-crystallin).

These observations confirmed that applying a low-molecular-weightchaperone provides benefits in terms of reducing mortality ratesfollowing serious burns.

Confirmation of Mortality Rate Reduction—Test 2

Next, mice that had received burns in the same way as in the precedingtests were used in tests to confirm mortality rate reductions dependingon the use or otherwise of a topical preventative medicament of thisembodiment against burns-related systemic inflammatory responsesyndrome, but to which a water-soluble fullerene had been added.

Specifically, a medicament was prepared combining 1% of water-solubleC60 fullerene (Vitamin C60 Co., Tokyo) with the topical medicament Aprepared in Test 1 for confirming mortality rate reductions (topicalmedicament B).

Tests were then carried out by the method used in Test 1 for confirmingmortality rate reductions. Three groups were established for the tests,namely, treated with topical medicament B, treated with topicalmedicament A, and untreated. These were compared ten hours afterreceiving the burns. The results of these tests are set out in FIG. 2.

As is clear from FIG. 2, survival was about 100% in the group treatedwith topical medicament B that had been mixed with a water-soluble C60fullerene to improve cell membrane penetrability (p<0.005), and thelevel was higher than in the group treated with topical medicament A.

This confirmed that the mortality rate reduction seen with alow-molecular-weight chaperone can be further improved by combining theuse of a water-soluble C60 fullerene.

Confirmation of Mortality Rate Reduction—Test 3

Next, mice that had received burns in the same way as in the twopreceding tests were used in tests to confirm mortality rate reductionsdepending on the use or otherwise of a topical preventative medicamentof this embodiment against burns-related systemic inflammatory responsesyndrome, but to which histamine had been added.

Specifically, a medicament was prepared adding histamine to a finalconcentration of 1 μM to topical medicament A prepared in Test 1 forconfirming mortality rate reductions (topical medicament C). As thecontrol, a medicament was prepared adding anti-histamine to a finalconcentration of 1 μM to topical medicament A (topical medicament D).

Tests were then carried out by the method used in Test 1 for confirmingmortality rate reductions. Two groups treated with topical medicament Cand with topical medicament D were established for the tests and theywere compared ten hours after receiving the burns.

Consequently, whereas no marked reduction in mortality rate was seen inthe group treated with topical medicament D compared to that treatedwith topical medicament A described earlier, the group treated withtopical medicament C showed a marked reduction in mortality ratecompared to those treated with topical medicament D or topicalmedicament A. It seemed that the life-prolonging action had beenincreased by histamine, which promotes vascular endothelialpermeability, and that it had conversely been decreased by adding ananti-histamine.

These findings served to confirm that the effect of topical medicament Ato reduce mortality rates could be improved still further by combiningthe use of histamine.

Tests to Confirm Suppression of Rises in TNF-α and White Blood CellCount

Rises in TNF-α and white blood cell count are generally seen in SIRSfollowing shock due to burns. Validation was carried out to confirm thatthese topical medicaments act to suppress rises in TNF-α and white bloodcell count following severe burns.

Two groups were established for these tests, one treated with topicalmedicament A (N=5) and another untreated group (N=5). TNF-α (pg/mL) andwhite blood cell count (cells/μL) were determined three hours after theburns had been received. An ELISA kit (Shibayagi, Japan) was used todetermine mouse TNF-α. The results are shown in FIG. 3 and FIG. 4.

As is clear from FIG. 3, the results of these tests demonstrated thatthe level of serum TNF-α was markedly suppressed by the application oftopical medicament A compared to no treatment.

As is clear also from FIG. 4, the white blood cell count was markedlycontrolled by the application of topical medicament A compared to notreatment.

It was evident from these findings that this topical medicament ishighly effective for suppressing the marked rises in TNF-α and whiteblood cell count seen after burns, and it was suggested that this couldsuppress burns-related SIRS and contribute to reduced mortality rates.

As stated above, because the topical preventative medicament of thepresent invention against burns-related systemic inflammatory responsesyndrome contains a low-molecular-weight chaperone as its main activeingredient, it is possible to provide a topical preventative medicamentagainst burns-related systemic inflammatory response syndrome capable ofsuppressing SIRS due to burns by applying it immediately to the affectedarea when someone suffers a burn.

Finally, the descriptions of each of the above embodiments are examplesof the present invention and the invention is not limited to theembodiments described above. Therefore various changes can be made asappropriate to the design and the like to produce embodiments other thaneach of those described above, as long as they do not deviate from thetechnical concept of the invention.

What is claimed is:
 1. A topical preventative medicament againstburns-related systemic inflammatory response syndrome containing alow-molecular-weight chaperone as the main active ingredient.
 2. Thetopical preventative medicament against burns-related systemicinflammatory response syndrome according to claim 1, characterized inthat it further contains a water-soluble fullerene.
 3. The topicalpreventative medicament against burns-related systemic inflammatoryresponse syndrome according to claim 1, characterized in that it furthercontains histamine.
 4. The topical preventative medicament againstburns-related systemic inflammatory response syndrome according to claim1, characterized in that the low-molecular-weight chaperone isαB-crystallin.
 5. A medicament comprising a topical preparationcontaining an effective amount of a low molecular-weight chaperone. 6.The medicament of claim 5, wherein the chaperone is a sHSP.
 7. Themedicament of claim 6, wherein the chaperone is αB-crystallin.
 8. Themedicament of claim 5, wherein the medicament is an aqueous solution. 9.The medicament of claim 8, wherein the aqueous solution is a bufferedsolution.
 10. The medicament of claim 8, further comprising one or moreof histamine and a soluble fullerene.
 11. The medicament of claim 8,wherein the chaperone is present at a concentration of 1-100 μM, 10-80μM or 25-75 μM.
 12. The medicament of claim 11, further comprisinghistamine present at a concentration of 0.05-1.0 μM, 0.05-0.5 μM or0.05-0.1 μM.
 13. A spray bottle containing a topical preparationcontaining an effective amount of a low molecular-weight chaperone. 14.The spray bottle of claim 13, wherein the chaperone is a sHSP.
 15. Thespray bottle of claim 13 wherein the chaperone is αB-crystallin.
 16. Thespray bottle of claim 13, wherein the medicament is an aqueous solution.17. The spray bottle of claim 16, wherein the aqueous solution is abuffered solution.
 18. The spray bottle of claim 16, further comprisingone or more of histamine and a soluble fullerene.
 19. The spray bottleof claim 16, wherein the chaperone is present at a concentration of1-100 μM, 10-80 μM or 25-75 μM.
 20. The spray bottle of claim 16,further comprising histamine present at a concentration of 0.05-1.0 μM,0.05-0.5 μM or 0.05-0.1 μM. 21.-28. (canceled)